Journal of Clinical Toxicology
Research Article
Alnuqaydan and Sanderson, J Clin Toxicol 2016,
6:5
DOI: 10.4172/2161-0495.1000316
OMICS International
Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care
Products on HaCaT Human Skin Cells
Abdullah M Alnuqaydan* and Barbara J Sanderson
Department of Medical Biotechnology, School of Medicine, Flinders University, Australia
*Corresponding
author: Abdullah M Alnuqaydan, Department of Medical Biotechnology, School of Medicine, Flinders University, GPO Box 2100, Adelaide, SA 5001,
Australia, Tel: +61-08-7221-8567; Fax: +61-08-7221-8555; E-mail: [email protected]
Received date: June 15, 2016; Accepted date: September 17, 2016; Published date: September 20, 2016
Copyright: © 2016 Alnuqaydan AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Human skin is normally exposed to ionizing and UV radiations and on occasions it may also be
subjected to beauty products or drugs that the host uses; all of which can generate reactive oxygen species (ROS).
Objective: In this study, Calendula officinalis extract, which contains antioxidant compounds, was examined for
its protective effects against the products that generate ROS- induced cytotoxic activity towards human skin cells.
Methodology: The protection against cytotoxicity by Calendula officinalis extract was investigated in vitro using a
dose-response on HaCaT human skin cells. The proprietary aqueous calendula extract C from biodynamically grown
plant was examined. Protection against cytotoxicity was measured via the methyl tetrazolium cytotoxicity (MTT)
assay. Cells were exposed to the calendula extracts for 48h before being exposed to personal care products
consisting of two head lice treatments -Lice Breaker (Permethrin 1% w/w) and Organix Pyrethrum treatment (4 g/L
Pyrethrins, 16 g/LPiperonyl Butoxide) and two beauty products -Nivea Visage Q10 plus Anti-Wrinkle face moisturizer
+ TiO2; and Nivea Visage Q10 Plus Anti-Wrinkle face moisturizer (without TiO2) for 1h.
Results: The effect of different concentrations of calendula extract on HaCaT human skin cells in vitro was
explored. Doses of 1% (v/v) [1.4 mg dry weight equiv/ml] or less showed no toxicity. Cells were also exposed to the
calendula extract for 48 hours before being exposed to the four personal care products for 1h. using the MTT
cytotoxicity assay; it was observed that extract of Calendula officinalis gave time-dependent and concentrationdependent protection against harmful products that induce cell killing. Pre-incubation with the calendula extract for
48 hours significantly increased survival relative to the population without the extract by 30% and 47.4%,
respectively, following treatment with personal care products.
Conclusion: This study demonstrates that calendula extract contains bioactive and free radical scavenging
compounds that significantly protect against personal care products that generate oxidative stress cytotoxicity in a
human skin cell culture model.
Keywords: Toxicity; Calendula officinalis; Beauty products; Lice
treatments; Cell culture
Introduction
Human skin exposed to ionizing and UV radiations or drugs and/or
personal care products such as head lice treatments or beauty products
that could generate reactive oxygen species (ROS) in vast quantities,
could oxidize degrading pathways [1]. Uncontrolled ROS can be
implicated in many aspects of pathogenesis such as human skin
disorders, for example cutaneous neoplasia [1,2]. In human skin there
are many agents that can produce oxidative stress including industrial
sources, UV radiation, food contaminants, additives, drugs or cosmetic
products [1,3]. Also, reactive nitrogen species (RNS) can be formed
from exposure to environmental agents like xenobiotics [1]. Oxidative
stress interacts with the process of variety of skin diseases [1].
Calendula flower has been shown to protect against cells being killed
and chromosomal damage induced by hydrogen peroxide (H2O2) in
HaCaT human skin cells [4,5]. It was shown that Calendula officinalis
contains a number of compounds with antioxidants and free radicals
J Clin Toxicol, an open access journal
ISSN:2161-0495
with scavenging potential. Also, Calendula plant was used as a skin
conditioning agent in cosmetics because of its anti-inflammatory
properties [6]. The other use of the Calendula plant includes treatment
for first degree burns and rashes [6]. However, chemical head lice
treatments and chemical beauty products can induce toxicity and
genotoxicity in human skin cells [7]. The hypothesis is that personal
care products such as chemical head lice treatments and beauty
products induce toxicity effects by producing oxidative stress in human
skin cells.
In this study, we examine two head lice treatments (Lice Breaker
treatment (Permethrin 1% w/w) and Organix Pyrethrum treatment (4
g/L Pyrethrins plus 16 g/L Piperonyl Butoxide)) and two beauty
products (Nivea Visage Q10 plus Anti-Wrinkle face moisturizer
(contains mixture of chemicals ingredients) + TiO2; and Nivea Visage
Q10 Plus Anti-Wrinkle face moisturizer (contains mixture of
chemicals ingredients) (without TiO2). The objective of this study is
that to determine the ability of the calendula flower extract to protect
human skin cells against any detrimental effects of these personal care
products on human skin cells in vitro.
Volume 6 • Issue 5 • 1000316
Citation:
Alnuqaydan AM, Sanderson BJ (2016) Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on
HaCaT Human Skin Cells. J Clin Toxicol 6: 316. doi:10.4172/2161-0495.1000316
Page 2 of 5
Material and Methods
Materials
1640 RPMI medium, foetal bovine serum (FBS) and penicillin/
streptomycin were purchased from Gibco, Invitrogen, Barcelona.
Phosphate buffered saline (PBS) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) were purchased from (Sigma™).
Media for growth and treatment were 1640 RPMI medium with 10%
FBS. All the reagents used in this study were purchased from SigmaAldrich unless otherwise stated.
Plant extract and characterization
The characterization of calendula extract C and its ability to protect
human skin cells HaCaT against hydrogen peroxide (H2O2) induced
oxidative stress cell killing and genetic damage was published
previously [4,5]. Briefly, dried flowers of Calendula officinalis were
supplied by an industry partner (Jurlique International, Mount Barker,
South Australia) from biodynamically grown plants. Calendula extract
C was prepared as a proprietary aqueous extract. It was diluted in
RPMI media to make the treatment solution. The determination of
density for extract C (1.036) was carried out at 20°C from the average
of 10 independent measurements. The dry weight of extract C was 1.4
mg/ml as determined after extracts samples were freeze dried at -56°C
at 3.4 Pa for 24h. The Folin–Ciocalteu assay was used to estimate
polyphenol composition and the gallic acid equivalent of extract C was
10.83 mg GAE/g dry weight. Also, DPPH assay was used in previously
published work to perform the free radical inhibition of extract C.
Extract C showed 47.7% radical inhibitions at concentration 2.5% (3.5
mg/ml dry weight equivalent).
h at 37ºC. Formazan crystals were then dissolved in SDS (20% sodium
dodecyl sulphate in 0.02 M HCL) for overnight. The absorbance was
measured on an ELISA plate reader with the background reference
wavelength of 630 nm and a test wavelength of 570 nm.
Statistical analysis
The experiments were done in triplicate and data is presented as the
mean ± S.E.M. One-way ANOVA with Tukey’s posthoc test was carried
out using SPSS software, version 22. Also, one-tailed t-test was also
used. Differences were considered to be statistically significant when
the P-value was less than 0.05.
Results
The toxicity of Calendula extract C on HaCaT cells in vitro was
explored by incubating cells with extract for 48 h. Figure 1 showed the
toxicity of extract C. calendulas extract C showed toxicity only at a
high dose (5%) for the 48h treatment time. At the lowest doses, the
extract (0.125, 0.5, 1.0 and 2.0 % (v/v)) was not toxic on HaCaT skin
cells for any of the treatment periods. This effect could involve a period
of contact between these components and the cells, such that these
toxic components would be taken up by cells, thus reducing the cellular
viability. Therefore, in contrast, lower concentrations of such extracts
would not be toxic if incorporated into products at a safe dose, which
is difficult to ascertain on human skin. The toxicity at the higher doses
of calendula extracts that has been found in this study needs to be
translated into a recommendation for use of calendula extract on
human skin.
Cell culture
A human non-cancer keratinocytes cell line HaCaT was like a gift
from the Department of Haematology & Genetic Pathology - Flinders
Medical Centre, School of Medicine at Flinders University, Adelaide,
South Australia. It was maintained in RPMI 1640 medium, with 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo
Scientific, Australia). Cells were seeded in tissue culture flasks and
incubated at 37°C in a 5% CO2 fully humidified incubator. HaCaT cells
were subcultured when they reached 60–80% confluence.
Cell treatment
HaCaT cells were treated to allow toxicity exposure before the
protection assay being conducted. Microplates 96 wells -flat bottom
were seeded with104 cells/well and incubated for 19 h to allow cells to
adhere at a temperature of 37°C in 5% CO2. The media were aspirated
and replaced with 200 µl of the treatment solution which was 0.125,
0.5, 1.0, 2.0 and 5% (v/v) extract C per well for 48 h.Then follow up
with 100 µl products treatments (head lice treatments or titanium
dioxide or beauty products) for 1 h before MTT assay analysis. The
negative or zero control was media.
MTT cell survival assay
To measure cell viability of HaCaT human skin, the methyl
tetrazolium cytotoxicity assay was performed as published [4,8,9].
Briefly, the treatment solution was aspirated, and wells washed twice
with PBS, cells were incubated with 200 µl MTT (0.5 mg/ml)/well for 4
J Clin Toxicol, an open access journal
ISSN:2161-0495
Figure 1: Relative survival at 48 h for calendula extract. HaCaT cells
were treated for the time indicated then assayed by MTT assay (see
section 2.4). Data are shown as mean survival relative to the
untreated control ± SEM; n=3. * = treatments are significantly
different from the 0 untreated controls at p<0.05.
Head lice treatments can induce cell killing act as apoptosis or
necrosis [7]. Figure 2 showed the toxicity results of personal care
products on HaCaT human skin cells. HaCaT human skin cells were
pre-treated for 48h with 1% calendula extract C then followed by 1 h
treatment with personal care products (beauty products or head lice
treatments). Permethrin doses of 10% and 100%, Pyrethrum doses of
10% and 50% and Nivea visage at doses 0.3% and 3% with or without
TiO2 proved to be significantly toxic in HaCaT cells when measured by
MTT assay.
Volume 6 • Issue 5 • 1000316
Citation:
Alnuqaydan AM, Sanderson BJ (2016) Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on
HaCaT Human Skin Cells. J Clin Toxicol 6: 316. doi:10.4172/2161-0495.1000316
Page 3 of 5
Figure 2: Toxicity and Calendula protection of personal care products on HaCaT human skin cells measured by MTT assay. Permethrin (low
dose; 10%, high dose; 100%), Pyrethrum (low dose; 10%, high dose; 50%), Nivea visage without TiO2 (low dose; 0.3%; high dose; 3%) and
Nivea visage with TiO2 (low dose; 0.3%; high dose 3%). Data shown as mean ± SEM, n=3. * = treatments are significantly different from the 0
untreated control at p<0.05. ¥= pre-treatment with calendula extract significantly different from products toxicity (protection).
The lower concentration of Calendula extract C 1% chosen as pretreatment for HaCaT cells due to its safety treatment for HaCaT cells
for all treatments period. Calendula officinalis extract showed the
ability to protect significantly against toxicity effects of personal care
products including TiO2 treatment after treated HaCaT cells with
Calendula extract C for 48h as illustrated in Figure 2. Calendula
treatment for 48h offers more protection than 24h treatment time
(Data not shown).
Toxicity for
Pre-treatment
calendula extract for
Sample
1h
48 h
Medium control
100 ± 1.3
100 ± 1.5
*67.8 + 1.4
73.6 ± 10.9
*58.2+3.2
**73.3 ± 5.2
with
TiO2 low dose
(150 µg/ml)
TiO2 high dose
(200 µg/ml)
Table 1: Untreated control and Titanium Dioxide (TiO2) the positive
control of beauty products. Data shown as mean ± SEM, n=3. * =
treatments are significantly different from the 0 untreated control at
J Clin Toxicol, an open access journal
ISSN:2161-0495
p<0.05. **= treatment is significantly different from TiO2 dose at
p<0.03 carried out using one- tailed T-Test.
Table 1 shows the untreated and positive controls of Titanium
dioxide TiO2 in the experiments. Cells were incubated for 48 h with
calendula extract C alone there was a mean increase of 62% in relative
survival. This is likely due to some cell growth during the incubation
period.
It is important that the protection observed for the personal care
products is real. The reason is that the positive control TiO2 challenge
still results in cells being killed and only a small level of protection. Of
note, the treatment of calendula alone lead to an increase in cell
number. This was not used as the untreated control as the correct
control was the untreated cells.
Discussion
The human body is exposed to variety of pro-oxidants in the
environment including drugs, chemicals, mixtures, radiation,
pollutants or cosmetics. These agents can target lipid-rich membranes,
cellular DNA or proteins to produce toxicity. Personal care products
such as beauty products can generate oxidative stress in human skin
cells [1]. The increases in ROS play a role in a variety of skin diseases
and carcinogenesis [1,10]. Beauty products and head lice treatments
Volume 6 • Issue 5 • 1000316
Citation:
Alnuqaydan AM, Sanderson BJ (2016) Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on
HaCaT Human Skin Cells. J Clin Toxicol 6: 316. doi:10.4172/2161-0495.1000316
Page 4 of 5
examined in this study induced significant toxicity in HaCaT human
skin cells. The hypothesis was that personal care products such as
beauty products or head lice treatments could generate oxidative stress
in human skin cells. As a result, there was significant toxicity induced
after treating HaCat cells with these products for 1h. The toxicity
results of personal care products on HaCaT cells were measured by
MTT assay which is a relative survival assay. Calendula officinalis
showed protection activity against hydrogen peroxide (H2O2) induced
oxidative stress cell killing and chromosomal damage [4,5]. Therefore,
HaCaT cells treated with Calendula extract C to produce bioactivity
antioxidant compounds did protect against toxicity of personal care
products (beauty products and head lice treatments) that caused
oxidative stress cell killing. The protection offered by Calendula extract
C was time-dependent as explored early [4,5]. It was noted that there
was significantly higher protection after pre-treatment with calendula
extract with compounds that displayed higher toxicity when induced
by the treatment of compounds alone. This was seen in the protection
with the high of Pyrethrum, Nivea visage and Nivea visage with TiO2.
Further work needs to be done to understand the mechanism of action
of calendula plant components and how they work to protect human
skin cells. TiO2 was classified as a carcinogenic nanoparticle used in
cosmetics. Titanium dioxide (TiO2) plays a role in the induction of
apoptosis as well as oxidative stress [11-13]. One study indicated that
Titanium dioxide TiO2 can affect the mitochondria [14].
Calendula extract contains bioactives and free radical scavenging
compounds that can interact with ROS to either eliminate them or
minimise their deleterious effects [1]. These antioxidants or bioactive
compounds including ascorbic acid, vitamin E, Vitamin C, alphatocopherol, quercetin, beta-carotene and amino acid [1,4]. Oxidative
stress also drives the production of oxidation products, e.g. 4hydroxy-2-nonenal or malonaldehyde that can denature proteins and
alter apoptosis or influence the release of pro-inflammatory mediators
such as cytokines in inflammatory skin diseases [1]. Moreover, ROS in
the induction of many biological responses can act as second
messengers such as in the generation of cytokines [15]. The alterations
in cellular proteins or peroxidation of lipid-rich membranes caused by
ROS can contribute to a range of skin diseases or cancer [1].
Experimental evidence supports the role played by ROS in the cancer
process [1,10].
The increases of ROS in the cell, through either endogenous or
exogenous factors, contribute to the carcinogenesis process. This could
occur via genotoxic effects resulting in oxidative DNA adducts or via
modification of gene expression [10]. MTT assay was employed in this
study as a colorimetric assay for mammalian cell growth and survival,
and it depends on the ability of viable cells to metabolize the yellow
and water-soluble tetrazolium salt in the mitochondria of living cells. It
can be used for mitochondrial dysfunction [9,16]. Mitochondrial
damage is linked to the induction of mutations [17]. Moreover,
mitochondrial DNA mutation and alteration in gene expression
(mutation in the gene encoding for complexes I, II, IV and V) has been
identified in many types of cancers and human tumours [17]. The
mutation rate in mitochondrial is reported to be greater than in
nuclear DNA [18].
In conclusion, it is evident that beauty products and head lice
treatments examined in this study induced significant toxicity on
HaCaT human skin cells, and that could be due to generating oxidative
stress in skin cells. This process contributes to skin disorders such as
inflammation and carcinogenesis or cancer. Also, the positive control
of TiO2 beauty product which is a carcinogen nanoparticle does play a
J Clin Toxicol, an open access journal
ISSN:2161-0495
role in the oxidative stress by damaging the mitochondria in the cell
[11-14]. Finally, this study indicated that Calendula officinalis extract
C does protect HaCaT human skin cells against the toxicity caused by
beauty products and head lice treatments. Further work could be done
to determine the potential protection of calendula extract against the
chromosome damage induced by personal care products also
fluorescent cell viability dyes to specify the damaged chromosome and
cancer type on Human skin cells.
Acknowledgments
We thank the Ministry of Education, Saudi Arabia, for partial
support of this project. Also, thanks to Professor Christopher Franco,
Head of Medical Biotechnology, School of Medicine, Flinders
University for reviewing the manuscript.
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Volume 6 • Issue 5 • 1000316
Citation:
Alnuqaydan AM, Sanderson BJ (2016) Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on
HaCaT Human Skin Cells. J Clin Toxicol 6: 316. doi:10.4172/2161-0495.1000316
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